Further, Aβ1-42 was correlated with psychometric scores (p < 0.001) and CT (p < 0.01). CT was thinner in the affected patients (p = 0.035). No differences were observed for APP-CTF and p-tau relative abundance in tears. Testing Aβ1-42 levels in tears seems to be a minimally invasive, cost-saving method for early detection and diagnosis of AD.We aimed to evaluate the diagnostic role of Alzheimer’s disease (AD) biomarkers in tears as well as their association with retinal and choroidal microstructures. In a cross-sectional study, 35 subjects (age 71.7 ± 6.9 years) were included: 11 with prodromal AD (MCI), 10 with mild-to-moderate AD, and 14 healthy controls. The diagnosis of AD and MCI was confirmed according to a complete neuropsychological evaluation and PET or MRI imaging. After tear sample collection, β-amyloid peptide Aβ1-42 concentration was analyzed using ELISA, whereas C-terminal fragments of the amyloid precursor protein (APP-CTF) and phosphorylated tau (p-tau) were assessed by Western blot. Retinal layers and choroidal thickness (CT) were acquired by spectral-domain optical coherence tomography (SD-OCT). Aβ1-42 levels in tears were able to detect both MCI and AD patients with a specificity of 93% and a sensitivity of 81% (AUC = 0.91). Tear levels of Aβ1-42 were lower, both in the MCI ( The identification of new, less invasive, and cost-saving methods for the early detection of Alzheimer’s disease (AD) currently represents one of the main challenges in research. At present, the most well-established biomarkers for AD include the evaluation of beta-amyloid peptides Aβ1-42), total tau (t-tau), and phosphorylated-tau (p-tau) in the cerebrospinal fluid (CSF) as well as the identification of amyloid deposition in the PET scans -42, tota,4,5. HowAccording to the inclusion and exclusion criteria, 66.1% of the subjects assessed for eligibility were enrolled: 25 patients with MCI due to AD or mild-to-moderate AD, and 16 healthy controls. After enrollment, 4 affected patients and 2 healthy controls were further excluded for insufficient tear sample collection. Finally, 11 patients with MCI, 10 patients with mild-to-moderate AD, and 14 healthy patients were included in this study. A detailed flowchart of patients’ enrollment and further exclusions is reported in Overall, as shown in The same characteristics were analyzed in the three groups of study subjects .Ophthalmological data were tested for the differences between affected patients and healthy subjects and are reported in p = 0.035, diff. 39.1, CI 3.0 to 75.3). In p = 0.069, contrast −49.4, CI −101.9 to 3.2) after one-way ANOVA  = 2.78, p = 0.077).As shown, in both groups, patients were mostly phakic and had a compatible refractive error (SEQ). Concerning SD-OCT findings, the analysis of pRNFL as well as central foveal thickness, NFL and GCP-IPL showed high compatibility between affected patients and controls. However, when comparing choroidal thickness (CT), affected patients showed a significant thinning compared to the control group  highlighted a substantial difference among the three groups. Specifically, Dunn’s pairwise comparison revealed that Aβ1-42 concentrations were significantly lower in MCI  and AD  groups when compared to the control group, while no differences were found between the MCI and AD groups . In Concerning the evaluation of Aβ1-42 by ELISA, a Kruskal–Wallis rank test (χ2(2) = 17.908, p = 0.257), no differences among the three groups  were highlighted. In With regard to the p-tau relative abundance as evaluated by Western blot, after the Kruskal–Wallis rank test (χ2(2) = 2.712, p = 0.002). A Tukey post hoc test highlighted that APP-CTF was lower in MCI patients when compared to AD patients. However, no significant differences were observed when comparing MCI patients and AD patients to the control group. In Regarding the estimation of APP-CTF fragments, Western blot evaluation revealed the presence of a predominant immunoreactive band, with a molecular weight of approximately 25 kDa. Notably, the detection of 25 kDa CTF fragments was already observed in CSF from AD patients . In thisRepresentative Western blot of p-tau and APP-CTF are shown in p < 0.001), MMSE , ADAS-Cog , and CT . A post hoc analysis (p = 0.015).When testing Spearman’s rank correlations, Aβ1-42 was found to be correlated with the affected group , corrected by age, sex, and all the tested AD biomarkers as well as CT, lower levels of Aβ1-42 were retained as the only predictor for MCI and AD .A receiver operating characteristic (ROC) curve  was run In the TEARAD study, AD biomarkers were tested in the tears of a cohort of 21 affected patients and 14 healthy subjects. Compared to the control group, both MCI due to AD  and AD patients showed lower tear levels of Aβ1-42 and its concentration turned out to be a predictor for the disease (AUC 90.65%). In addition, affected patients showed lower CT and this turned out to be directly correlated with Aβ1-42 levels. To our knowledge, this is the first study analyzing Aβ1-42, APP-CTF, and p-tau in tear samples of a cohort of fully characterized AD patients according to the NIA-AA criteria and comparing biomarkers’ concentrations with an age- and sex-matched control group selected according to strict systemic and ocular inclusion criteria.The idea to test AD biomarkers in tears was proposed and evaluated by a few authors without conclusive results or practical outcomes. Previous studies on AD patients’ tears are hardly comparable as they differ in patients’ baseline characteristics , tear collection procedure, evaluated biomarkers, as well as molecular analysis techniques ,10. KallIn the literature, there is only one study that investigated the presence of AD typical biomarkers in the tears of a cohort of affected patients . SpecifiIn our findings, significantly low tear levels of Aβ1-42 seem to be consistent with the reported pattern of biomarkers’ levels in CSF. Reduced levels of Aβ1-42 in the CSF are considered to be a result of the sequestration of peptide in the brain ,17. ThusRegarding APP-CTF levels in tears, we failed to find substantial differences between patients and controls. Similarly, we did not observe any significant change in p-tau levels. Despite this, we succeeded in detecting the presence of p-tau in the tears of all included participants. This is sharply in contrast to the work from Gijs and colleagues, who did not detect p-tau in healthy controls and detected it only in 27% of demented patients. To overcome technical issues, the authors encourage the employment of other tests with higher sensitivity to detect p-tau. It is probable that the ultra-sensitive ECL reagents, as well as the different methodologies employed in our study, may have improved the ability to detect the protein in tear samples. Despite this consideration, further studies are required to confirm these findings by analyzing different techniques in parallel.Concerning the ophthalmologic findings of our study, a reduction of CT was found in patients compared with the controls. A thinner choroid was already described in AD patients and could be associated with hypoperfusion and/or atrophic changes in the choroid, but this is the first study to explore a correlation between a biomarker in tears and the choroid . In partAmong the strength of this study, it should be mentioned that there was a strict protocol of inclusion and exclusion criteria with well-defined criteria for MCI and AD diagnosis, age- and sex-matched population for the healthy controls, and the employment of a tear collection method as well as ELISA analysis for Aβ1-42 levels in tears that avoided excessive dilution of tear samples or normalizing formulas. Among the shortcomings, the small sample size, the non-availability of biomarkers’ analysis in the CSF, and the absence of a control group with non-AD-related dementia should be disclosed.Overall, the results of the TEARAD study suggest that lower levels of Aβ1-42 in tears could be strongly associated with AD, pointing the way to a new promising, minimally invasive, and cost-saving method for early detection and diagnosis of AD. These findings justify future larger trials and provide useful preliminary data to plan future prospective studies to confirm the diagnostic role of Aβ1-42 in tears.A non-pharmacological cross-sectional cohort study was conducted at the Policlinico Umberto I University Hospital of Rome from September 2020 to December 2021. The study, aiming to identify AD biomarkers in human tears (TEARAD), was performed in adherence to the tenets of the Declaration of Helsinki and was approved by the ethical board of the Sapienza University of Rome . Forty right-handed patients with mild cognitive impairment (MCI) due to AD  and mild-to-moderate AD attending the Department of Neurosciences and Mental Health of the Policlinico Umberto I University Hospital of Rome were assessed for eligibility to participate in the study. Parallelly, twenty age- and sex-matched volunteers with no cognitive disorders were consecutively assessed for recruitment among patients’ partners or caregivers. At the time of enrollment, all participants underwent physical and neurological examination and standard laboratory tests including serum folate, vitamin B12, and thyroid hormone assays.General inclusion criteria for patients and controls were age between 50 and 85 years at the time of enrollment and written informed consent to the study signed by patients. To be included in this study, all the patients with prodromal AD had to meet the NIA-AA core clinical criteria for MCI due to AD, have a Clinical Dementia Rating Scale (CDR) score of 0.5 (memory box score of 0.5 or greater at screening) and a Mini-Mental State Examination (MMSE) score >24. All the participants with mild-to-moderate AD were enrolled if they met the NIA-AA core clinical criteria for probable AD, had a CDR score of 1–2 with a memory box score of at least 0.5, and a MMSE between 15 and 26 . All patAll participants underwent a complete ophthalmological evaluation that included medical and ophthalmic history, spherical equivalent (SEQ), lens status, IOP measured with Goldmann applanation tonometry, and anterior segment as well as fundus evaluation. According to a previously described protocol, SD-OCT  macular scans were automatically segmented to measure retinal thicknesses of the nerve fiber layer (NFL) and ganglion cell layer (GCL) together with inner plexiform layer (IPL) . Two bliAccording to a previously published protocol, tear samples were collected from affected patients and controls by microsponges applied to the lower eyelid margin of both eyes, centrifuged at 10,000 rpm per 10 min. Centrifugated tears from both eyes were pooled together and stored at −80 °C until evaluation . C-termiHuman Aβ1-42 levels in tear samples were assessed by using Human Aβ1-42 ELISA Kit with analytical sensitivity <10 pg/mL , according to the manufacturer’s instructions. To assess the concentration of p-tau and APP-CTF, tear samples were prepared for Western blot analysis. To start, the method of Lowry was employed to quantify protein concentration in each sample. Subsequently, Laemmli buffer was added, and samples were denatured at 95 °C for 3 min. Protein extracts (forty micrograms of proteins) were resolved on SDS-PAGE and transferred onto nitrocellulose membrane by using a trans-blot turbo transfer system . Thereafter, the membrane was incubated at room temperature for 1 h with 5% no-fat dry milk in Tris-buffered saline  and probed overnight at 4 °C with the following primary antibodies: anti-tau phospho T181 ; anti-β-Amyloid . Membranes were successively incubated for 1 h at room temperature with HRP-conjugated secondary anti-rabbit or anti-mouse antibodies . Protein-bound antibodies were visualized by Immobilon ECL Ultra Western HRP Substrate , and chemiluminescence was registered through the ChemiDoc MP system . Densitometric analysis derived from Western blots was then carried out by using ImageJ version 1.52t  software for Windows. Densitometric calculations were obtained as arbitrary units (a.u.), derived from the ratio between the intensity of the protein band and the respective Ponceau S band, used as the loading control.We based sample size calculation on the Aβ1-42 levels in tears, as it was the only biomarker that we assessed with a quantitative method (ELISA). Since no conclusive primary data regarding Aβ1-42 levels in tears was available, we assumed Aβ1-42 levels in tears to be specular to Aβ1-42 levels in CSF and 10 times less concentrated as suggested by the previous literature ,15. EstiData were first analyzed by comparing the group of affected patients (MCI and AD) with that of healthy subjects to evaluate the role of the studied biomarkers to detect the affected patients. Further, to assess the ability of the potential biomarkers to discriminate the disease severity, we stratified data evaluating the differences among the three groups of enrolled patients: MCI, AD, and healthy subjects. To avoid inter-eye correlation, only one randomly selected eye of each patient was included in the statistical analysis of the ophthalmological data .p-values were reported. The normal distribution of continuous variables was checked with the Shapiro–Wilk normality test. To test for the difference between parametric values, the unpaired t-test was employed to compare the affected patients and the control group, and the one-way ANOVA to compare three groups: patients with MCI, mild-to-moderate AD, and the control group. After ANOVA, a Tukey post hoc test was run to perform pairwise comparisons of means with equal variances between groups. To test for the differences of the non-parametric variables, the Wilcoxon rank-sum test or the Kruskal–Wallis rank test were employed accordingly. After the Kruskal–Wallis test, a Dunn’s pairwise comparison with Bonferroni adjustment was performed. Fisher’s exact test was used to compare categorical variables, and counts and percentages were reported. For parametric values, bivariate relationships were evaluated by the Pearson coefficient, while for non-parametric values, Spearman’s rank correlation was employed. To identify factors predicting AD, different from neurological scores, a logistic regression analysis was run, including sex, age, p-tau, APP-CTF, Aβ1-42, and all ophthalmological or clinical parameters that showed to be different among groups. A receiver operating characteristic (ROC) curve was generated to test the ability of the identified factors to detect AD.Statistical analysis and graph generation were performed with STATA v. 17.0 . When appropriate, confidence intervals (CI 95%), interquartile range (IQR), and The availability of non-invasive and inexpensive methods for an early diagnosis and/or prognosis of AD will significantly impact patients’ quality of life and health perspectives. Low levels of Aβ1-42 may represent a specific, sensitive, non-invasive, and inexpensive biomarker of AD. Given the low levels of Aβ1-42 also in the MCI, this biomarker could also represent a potential tool for early diagnosis. The direct correlation between Aβ1-42 and CT seems to strengthen the hypothesis of a direct connection between the eye and the brain. Further studies with larger sample sizes, possibly correlating tear findings to CSF and PET or MRI imaging are needed to validate our results.


Rubber materials are widely used in aerospace, automotive, smart devices and artificial skin. It is significant to address the aging susceptibility of conventional vulcanized rubber and to impart it rapid self-healing performance for destructive crack damage. Herein, a novel supramolecular rubber elastomer is prepared by introducing metal coordination between carboxyl-terminated polybutadiene and polystyrene-vinylpyridine copolymer. Based on the metal coordination interaction, the elastomer exhibits shape memory and self-healing properties. Moreover, a rapid closure-repair process of destructive cracks is achieved by presetting temporary shapes. This shape memory-assisted self-repair model is shown to be an effective means for rapid repair of severe cracks. An approach to enhance the mechanical and self-healing properties of elastomer was demonstrated by adding appropriate amounts of oxidized carbon nano-onions (O-CNO) into the system. The tensile strength of the elastomer with an O-CNOs content of 0.5 wt% was restored to 83 ± 10% of the original sample after being repaired at 85 °C for 6 h. This study confirms that metal coordination interaction is an effective method for designing shape memory self-healing rubber elastomer. The shape memory-assisted self-healing effect provides a reference for the rapid self-repairing of severe cracks. Rubber has been widely used in aerospace, automotive, smart devices and artificial skin due to its excellent performance due to its high elasticity and high wear resistance. The traditional rubber generally needed to undergo vulcanization is processed to cross-link the raw rubber molecules to form a 3D network to optimize its performance. However, a stable and an irreversible covalent cross-linked structure makes the rubber difficult to recycle once it is aged. Therefore, developing a new elastomer molecular network construction approach to manufacture rubber materials with excellent mechanical properties and self-healing efficiency remains a challenge ,2,3.Supramolecular interactions include hydrogen bonding interaction, metal coordination interaction, π–π stacking interaction, host–guest interaction and other intermolecular interactions ,5. UrbanLiu  et al. cHowever, the properties of these self-healing elastomers are relatively single, generally showing good mechanical properties and self-healing properties. Here, we consider introducing metal coordination bonds into the block copolymer network, using the microphase separation properties of block copolymers and the metal coordination of supramolecular polymers to make the polymers form metal coordination block copolymer micelles ,15,16,17Moreover, carbon nano-onions (CNOs) with unique structural properties are typical 0D nanomaterials with high porosity, high electrical conductivity, and electrochemical stability ,19,20. SIn this paper, a self-healing polybutadiene-polystyrene-vinylpyridine elastomer based on metal coordination bond interaction [Zn(II)-carboxyl group and Zn(II)-pyridine] was prepared using polybutadiene rubber as the base material. An appropriate amount of O-CNOs was added to the elastomer to enhance its mechanical properties and self-healing ability. The well-dispersed O-CNOs reduced the intermolecular chain interaction forces, and the polymer chain mobility was enhanced, thus improving the self-healing ability. Moreover, the functionalized O-CNOs provide more abundant carboxyl groups, which enhanced the internal hydrogen bonding to improve the mechanical properties. The shape memory-assisted self-repair model was proposed as an effective means for rapid repair of large cracks. This multifunctional material integrating shape memory, self-healing, and shape-memory-assisted self-repairing properties will play a unique role in artificial muscles, artificial skin, intelligent control, sensing and other fields.Carboxyl-terminated polybutadiene  was purchased from Tianjin Xiens Biochemical Technology Co., Ltd. . The polystyrene-vinylpyridine (PS-b-P4VP) was purchased from Scientific Polymer Products; molecular weight was 400,000, and the content of pyridine functional group was 50 mol%. Toluene, ethanol and zinc acetate dihydrate were purchased from Aladdin Chemical Reagent Company. Carbon nano-onions  were purchased from Nanjing Mingchang New Material Technology Co., Ltd. . All chemicals were commercially available and used as received.2+, COO- and VP of 1:2:2, 1:2:4 and 1:2:8 were denoted as R1, R2 and R3, respectively.The compositions of all samples are listed in The CNOs and concentrated sulfuric acid were mixed to prepare a suspension; under the condition of constant stirring, concentrated sulfuric acid and concentrated nitric acid mixture with volume ratio of 3:5 was added to the above suspension and then cooled in an ice-water bath for 30 min. The mixture was then heated at 80 °C for 30 min to obtain a dispersion. Distilled water was added to the dispersion to dilute the dispersion; the dilution was centrifuged at 7500 rpm after standing for 12 h, and the dilution and centrifugation process was repeated twice. The black solid was collected to obtain O-CNOs powder.2+, COO− and VP in the prepared composite samples was 1:2:4, which were denoted as R2-0.5%, R2-1% and R2-2%, respectively, according to the mass ratio of their O-CNOs.O-CNOs powder and zinc acetate were dispersed in water/ethanol solution. PB-COOH and zinc acetate were dissolved in toluene solution. Mix the above two solutions well and let stand. After the solution was separated, PS-b-P4VP/toluene solution was added to it, and finally a gray mixed solution was formed. Use a rotary evaporator to remove most of the solvent from the grey mixed solution. The mixture was dried in a vacuum oven for 24 h to remove residual solvent, and a gray-brown translucent composite material was obtained. The molar ratio of Zn−1, and the transmission mode was used.Chemical bonds and functional groups of the samples were detected by Fourier Transform Infrared Spectroscopy (FT-IR) instrument . The number of scans was 32 times/s. The test range was 400–4000 cmThe molecular structure of the material was characterized by the laser microscope Raman spectrometer . The wavelength was 532 nm, and the light intensity was 10%.−1 and an argon atmosphere. The data reported are from measurements made after first preheating the samples to 320 °C and then cooling them to 40 °C to eliminate the influence of the prior thermal history.The thermal properties of the samples were measured with a Netzsch Instruments FTA449F5 differential scanning calorimetry (DSC) from 40 to 240 °C using a heating and cooling rate of 10 °C min−1. The tensile test uses dumbbell-shaped standard samples, and each specimen is tested more than five times in the same test environment to obtain the average value.The quasi-static tensile test was performed on a Instron 5967 universal tensile testing machine with a stretching rate of 10 mm minW of the elastic body is obtained by calculating the integral area enclosed by the hysteresis curve by the following formula:Cyclic tensile tests were performed on the samples at room temperature, the specimens were stretched to 50% strain, fully unloaded, and so on, and the hysteresis curves were measured at different cycles. The dissipated energy ΔAnalysis of self-healing performance: The wafer sample was cut into two semicircles with a blade and then healed at a certain temperature. The morphology of the sample before and after repair was observed by an optical digital microscope .−1. The self-healing efficiency was quantitatively evaluated according to the ratio of the tensile strength and elongation at break of the healed material to the original material, respectively.Dumbbell-shaped elastomer samples were cut in half from the middle with a blade; the sections were aligned at room temperature, pressed gently for 1 min and then placed in a vacuum drying oven at room temperature and 85 °C for healing. After the repaired samples were taken out from the vacuum drying box, they were cooled to room temperature and then subjected to stress–strain testing at a tensile rate of 10 mm × min−1, 1599 cm−1 to 1358 cm−1, 1608 cm−1, respectively. The ratio of intensities of the D and G bands (R = ID/IG) directly were related to the oxidation degree of the CNOs [2 by the powder sample. The crystallinity of the CNOs and O-CNOs were studied by XRD  of free carboxyl groups, respectively. It can be seen that the peak of PB-COOZn at 1740 cm−1 disappeared, instead, a broader peak appeared at 1580 cm−1, which confirmed the successful bonding of PB-COOH to Zn2+. For PS-b-P4VP, the peaks at 1413 cm−1, 1491 cm−1, 1556 cm−1, and 1595 cm−1 corresponded to the aromatic vibration of the pyridine ring, and the peak at 1595 cm−1 corresponded to the carbon-nitrogen bond (C-N) vibration. The peak of R2 and R2-0.5% appear at 1617 cm−1 next to 1595 cm−1, revealing the complexation of Zn2+ with the N atom of pyridine on PS-b-P4VP [−1 still existed, indicating that the complexation of Zn2+ with the carboxyl groups on the carboxyl-terminated polybutadiene was not disrupted. There was a clear relationship between the vasym(COO−) and vsym(COO−) bands in the FTIR spectra (region of 1300–1750 cm−1) and the metal-carboxylate coordination type. The separation of the bands (Δva-s = vasym − vsym) can indicate of the structure of carboxylate [a-s for Zn2+ was almost equal than that of Na+, revealing that coordination mode in PB-COOZn was bidentate bridging  to 0.83 ± 0.01 MPa (R2-2%) b,d,e. HoThe scanning electron microscope (SEM) was used to study the dispersion of O-CNOs in composite to illustrate the good mechanical properties. As shown in 2+ was 104–134 kJ/mol [2+ was approximately 250 kJ/mol [4 kJ/mol , and tha0 kJ/mol . The Zn(−1. To investigate the self-healing properties of the R2-0.5%, the original composite was cut into two separate samples using a blade. The two sections formed after cutting were aligned and pressed gently for 1 min, which were transferred to a vacuum drying oven at 85 °C for healing. It can be seen that the fracture interface of the composite was completely healed and the crack disappeared a,b. Likeg value of the R2 and thus enhanced the mobility of the polymer chains, which improved the self-healing properties of composite.The essence of the excellent self-healing ability of composite was further explained by the supramolecular interaction. g of the composite and enhanced the polymer chain flowability.Note that the self-healing effect of R2 and R2-0.5 can also occur at room temperature. As shown in 2+ acted as bridges to connect them, which allowed the system to generate shape memory effect.Healing of polymeric materials included crack closure and crack healing. Crack closure was essential for subsequent crack healing. The damage of materials was often accompanied by large deformations or cracks inside the materials in practical applications, which made it difficult for the material to self-repair without external force intervention. To address this challenge, the shape memory effect was proposed to rapidly close the crack to facilitate the self-healing process . Herein,Before that, the shape fixation rate and recovery rate before and after the addition of O-CNOs were calculated by testing the length of the original sample, the sample stretched to the elongation state, and the sample with shape recovery. The shape fixation rates of R2 and R2-0.5 were ~96% and ~93%, respectively, while their shape recovery rates were ~75% and ~64%, respectively. The addition of O-CNOs slightly led to the decrease of shape fixation and recovery rate of the composites, which may be attributed to the friction resistance produced by O-CNOs.Specifically, two samples with the same ratio as R2 were casted as U-shape and rectangular strip, and denoted as R2U and R2S, respectively. The rectangular strip sample R2S would endure a shape change and recovery process during which R2S was firstly bent into U-shape after being thoroughly heated at 60 °C and then cooled to fix the temporary U-shape. R2U and R2S, both in the U-shape state, were then scratched with a blade and simultaneously placed in a temperature environment of 60 °C a. As shoIn this paper, the elastomer with good shape memory effect, self-healing ability and mechanical properties was designed by introducing metal coordination and nano-filler into the polybutadiene rubber/polystyrene-vinylpyridine copolymer. The result indicated that the elastomer can be self-repaired at 85 °C for 6 h, and its repair efficiency can reach 83 ± 10%. The well-dispersed O-CNOs reduced the intermolecular chain interaction forces, and the polymer chain mobility was enhanced, thus improving the self-healing ability. Moreover, the functionalized O-CNOs provide more abundant carboxyl groups, which enhanced the internal hydrogen bonding to improve the mechanical properties. The as-prepared polymer also shows good shape memory assisted self-healing performance. This unique molecular network design not only provided a new network construction approach for the development of new rubbers to replace vulcanization but also blazed the way for the manufacturing of advanced elastomers with both excellent mechanical properties and self-healing ability.


The pigs were allocated to combined C5 and CD14 inhibition therapy group (n=4) and control group (n=8). The therapy group received intravenously C5 inhibitor (RA101295) and anti-CD14 antibody (rMil2) 30 min post-trauma. Controls received saline. Combined C5 and CD14 inhibition reduced the blood levels of the terminal complement complex (TCC) by 70% (p=0.004), CRP by 28% (p=0.004), and IL-6 by 52% (p=0.048). The inhibition therapy prevented the platelet consumption by 18% and TAT formation by 77% (p=0.008). Moreover, the norepinephrine requirements in the treated group were reduced by 88%. The inhibition therapy limited the organ damage, thereby reducing the blood lipase values by 50% (p=0.028), LDH by 30% (p=0.004), AST by 33%, and NGAL by 30%. Immunofluorescent analysis of the lung tissue revealed C5b-9 deposition on blood vessels in five from the untreated, and in none of the treated animals. In kidney and liver, the C5b-9 deposition was similarly detected mainly the untreated as compared to the treated animals. Combined C5 and CD14 inhibition limited the inflammatory response, the organ damage, and reduced the catecholamine requirements after experimental polytrauma and might be a promising therapeutic approach.Dysfunctional complement activation and Toll-like receptor signaling immediately after trauma are associated with development of trauma-induced coagulopathy and multiple organ dysfunction syndrome. We assessed the efficacy of the combined inhibition therapy of complement factor C5 and the TLR co-receptor CD14 on thrombo-inflammation and organ damage in an exploratory 72-h polytrauma porcine model, conducted under standard surgical and intensive care management procedures. Twelve male pigs were subjected to polytrauma, followed by resuscitation (ATLS Severely injured patients frequently develop complications, including coagulopathy, sepsis and multiple organ dysfunction syndrome (MODS) . The undThe complement cascade is activated within minutes after trauma , and rea+ cells  and damage-associated molecular patterns (DAMPs) recognition . In a re+ cells . In trau+ cells . Further+ cells , 13.+ cells was shown to distinctly change its transcriptional state in bacterial sepsis . Throughout 72-h of experiment, the animals were given fluids  at a rate of 0.5-2.0 ml/kg/h and parenteral nutrition  370 kcal/l 50-70 ml/kg body weight and day, under a close monitoring of the fluid balance. The heart rate and MAP were monitored using a Philips patient monitor . The femur fracture was reduced and fixed by an intramedullary nailing technique . For supporting the respiratory mechanics of pressure-controlled ventilated pigs, the animals were turned every 4-6 hours. If required, norepinephrine was administered i.v. for maintaining the MAP>60 mmHg.Twelve German landrace male pigs  from a disease-free barrier breeding facility were housed in ventilated rooms and allowed to acclimatize to their surroundings for a minimum of 7 days before surgery. 12-h before the experiment, the animals were exposed to fasting with water t et al. . Brieflyin vitro whole blood studies and in vivo mice studies showed the superiority of the combined inhibition therapy over the monotherapy  was provided by UCB Pharma . RA101295 is a C5 inhibitor, with a cross-species activity, that prevents C5 cleavage and the subsequent generation of C5a and formation of the terminal C5b–9 complement complex (TCC), which is present in two forms: as sC5b–9 in the fluid-phase and as the membrane attack complex (MAC) on the cell surface .rMil2 is a recombinant anti-porcine CD14 antibody , made recombinant as an IgG2/4 chimera in the laboratory of Professor T.E. Mollnes (Norway)  and prodThe pharmacodynamics of the combined inhibition therapy were assessed every 12-h. The RA101295 pharmacological effect of blocking the classical and alternative complement pathways was evaluated using the Complement System Screen Wieslab  according to the manufacturer’s instructions. CD14 antigen saturation with the rMil2 anti-CD14 antibody was assessed by flow cytometry analysis using FITC-labelled rMil2 anti-CD14 antibody. Briefly, 75µl of EDTA blood was incubated with anti-CD14-FITC antibody at a concentration of 375 µg/ml. The cells were analyzed using BD FACSCanto™ II. The data are presented as percentage of CD14-FITC positive cells (TCC assessment in EDTA-plasma was performed using the ELISA-based technique (lower detection limit: 0.156 CAU/ml) as previously described . BrieflyComplete blood count for the assessment of the white blood cells (WBCs) and platelets (PLTs) was performed using hematology analyzer . C-reactive protein (CRP), lipase, lactate dehydrogenase (LDH), and aspartate transaminase (AST) analyses were measured in serum by chemistry analyzer .The immunoassays for these analyses were chosen based on previous assessment of the different commercially available kits . Porcine® 568  was used as secondary Ab. The F-actin was stained using Phalloidin Alexa Fluor® 488 (Invitrogen). The slides were mounted with Vectashield mounting medium  containing DAPI for nuclear counterstain. The images were captured using a Zeiss Axio-Imager microscope . The images were analyzed based on the presence of absence of the fluorescent signal.Tissue-Tek OCT  embedded lung, kidney, and liver cryosections (7µm) were incubated overnight at 4°C with primary antibody: mouse monoclonal anti-C9 neoepitope exposed in C5b-9, clone aE11 as described above (provided by Professor T.E. Mollnes (Norway)). Goat anti-mouse IgG2a conjugated with Alexa Fluorwww.r-project.org) were used for data visualization and statistical analysis. The latter was primarily based on non-parametric approaches because of the low sample sizes. Efficacy of the interventional drugs was evaluated by means of different ancillary approaches:The results from Wieslab Complement System Screening and Flow Cytometry analysis are given as individual profiles (n=3). GraphPad Prism version 9.2.0  and the R software version 4.1.2 . The nonparametric Mann-Whitney-U test was used in order to compare treated and untreated groups at different time points. Further, to account for the repeated measurement structure of the data, a nonparametric approach of longitudinal data analysis in factorial designs was applied (R package nparLD) assessing the difference between treated and untreated animals (group changes over time) for the TCC, lipase, TNF, and NGAL data. Due to missing values, for the remaining variables  a mixed linear model analysis was applied, since the nparLD approach mentioned above requires complete data.On the other hand, for each individual trajectory an area under the curve (AUC) was calculated. These individual AUCs reflect the development of each particular outcome parameter over time. All individual AUC values were then summarized using median (IQR) for each group of treated and untreated animals separately. Again, the Mann-Whitney-U test was used to compare the median AUCs between treated and untreated animals. The percentage change of the AUC, reported in the text, represents the difference between the AUC median values of the untreated and the treated group. These AUC results are thought to support the findings from longitudinal data analysis. In the AUC graph representation, the treated pig who received lower medication dosages is depicted as filled black symbol color.Correlation analysis was performed using nonparametric Spearman rank correlation. For statistical analysis of the immunofluorescence staining results Fisher’s exact test was used. Statistical significance was assumed in case of a two-sided P-value < 0.05 . Because of the study setting, all p-values are interpreted in an exploratory manner, which is why there was no correction of the type 1 error level due to multiple hypothesis testing.One animal from the untreated group died at 60-h. It was included in the study analysis, while for the time point of 72-h the median of the other seven animals from the untreated group was calculated and used for the statistical analysis.The C5 inhibitor RA101295 injected i.v. 30 min post-injury as a bolus (3 mg/kg) with a subsequent continuous i.v. administration (0.55 mg/kg/h) until 64-h post-injury, blocked the classical complement pathway by 76% and the alternative pathway by 62% throughout the experiment (Intravenous administration of a single bolus of the anti-CD14 antibody (5mg/kg) 30 min after trauma ensured a saturation of 79% of the CD14 antigen on the granulocytes throughout the experiment  and a significant reduction of PLT consumption at the 72-h timepoint (p=0.026) in comparison with the untreated group  in both groups    (NCT02246595) and of an MD2-TLR4 antagonist  in severe sepsis (NCT00334828), but without any clinical effect. We have shown in an and TLR2 .+CD115+Ly6Chigh, human CD14+CD16-HLA-DRmid)  , 47, and stimuli . Mechani stimuli , 50. Com stimuli , 43, thu stimuli , was sigis model . Using sis model  adaptor is model . As CD14nhibitor . Thus, dBeyond sepsis research, complement antagonists are considered potential therapeutic strategies in severe cases of Covid-19 infections , 55. Thein vitro study using whole human blood incubated with bacteria and inhibition of complement and CD14 showed that post-challenge inhibition as compared to pre-challenge also attenuated the inflammatory response reasonably well during the first period of time, but the effect was gradually lost , but could be accompanied with a potential risk for statistical errors. Nevertheless, this did not weaken the robustness of the data, as the difference between the groups was substantial. The unequal sample sizes, due to drug supply shortages, are a limitation of the study. Nevertheless, it was carefully approached in the statistical analysis of the data. Because of the real-time pharmacodynamic analysis, the investigators could not be blinded, and due to drug availability issues, the animals could not be randomized. The performed meticulous monitoring of the pharmacodynamics of C5 and CD14 inhibitors allowed a prompt adjustment of the dosages for accomplishing the study goals. Although the first treatment animal received a lower dosage of the C5 and CD14 inhibitors, its pharmacological efficacy was comparable with the adjusted therapy regimen during the experiments, which included higher dosages of inhibitors and these four animals could therefore be combined as the treatment group. These findings justify the patient-tailored therapeutic approaches. Because the immune response of a multiple trauma patient is unpredictable, it requires close continuous monitoring, with a consequent adjustment of the therapy. The fact that most of the readouts were statistically significant with only four pigs in the treatment group and eight in the control group, underscores the substantial significance between the groups and that type I errors are highly unlikely. However, we cannot exclude that there were some type II errors.In summary, we demonstrated that the early administration of the combined blockade of C5 and CD14 post-injury, adjusted in response to subsequent immune monitoring, significantly reduced the catecholamine requirements, thrombo-inflammatory response, and organ damage in a 72-h porcine polytrauma model. These data have important implications for future therapeutic strategies in trauma.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Ministry for Nature, Environment and Consumer Protection in North Rhine-Westphalia, Recklinghausen, Germany; AZ 81-02.04. 2020.A215.MH-L, TM, FH, JG, MG, and KH designed the animal study. JG, ÜM, and LL performed the animal study. JG, ÜM, LL, KQ, XZ, AP, and YL collected the data. LL, JL, KP, and CL performed the experiments. BM, TM, and LL analyzed and interpreted the data. LL drafted the manuscript. All authors critically revised the manuscript and approved the final version.This research was funded by the Deutsche Forschungsgemeinschaft  – 429837092. RA101295 was provided by UCB Pharma .The authors would like to thank the Flow Cytometry core facility of University Clinic Aachen, especially Nathalie Steinke and Silke Vaßen for valuable support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Diabetic retinopathy is a frequent complication of diabetes mellitus and a leading cause of blindness in adults. The objective of this study was to elucidate the diabetic retinopathy pathophysiology in more detail by comparing protein alterations in human vitreous of different diabetic retinopathy stages.Vitreous samples were obtained from 116 patients undergoing pars plana vitrectomy. Quantitative immunoassays were performed of angiogenic factors  as well as cytokines  in samples from control patients  as well as diabetes mellitus patients without retinopathy (n = 25), non-proliferative diabetic retinopathy (n = 12), and proliferative diabetic retinopathy patients (n = 21). In addition, correlation analysis of protein levels in vitreous samples and fasting glucose values of these patients as well as correlation analyses of protein levels and VEGF-A were performed.We detected up-regulated levels of VEGF-A (p = 0.001), PIGF (p<0.001), Angiopoietin-1 (p = 0.005), Angiopoietin-2 (p<0.001), IL-1β (p = 0.012), and IL-8 (p = 0.018) in proliferative diabetic retinopathy samples. Interestingly, we found a strong positive correlation between Angiopoietin-2 and VEGF-A levels as well as a positive correlation between Angiopoietin-1 and VEGF-A.This indicated that further angiogenic factors, besides VEGF, but also pro-inflammatory cytokines are involved in disease progression and development of proliferative diabetic retinopathy. In contrast, factors other than angiogenic factors seem to play a crucial role in non-proliferative diabetic retinopathy development. A detailed breakdown of the pathophysiology contributes to future detection and treatment of the disease. Diabetic retinopathy (DR) occurs as a secondary disease of diabetes mellitus (DM), affects adults aged 20–74 in developed countries, and is one of the most common causes for new cases of blindness . As a reDR can be subdivided into different stages, starting with the early stage of mild non-proliferative DR (NPDR) and further progressing to the proliferative DR (PDR) stage. NPDR is mainly characterized by vascular changes and circulatory defects, whereas PDR is characterized by pathological preretinal neovascularization , 4. BaseAs in other neurodegenerative diseases, inflammation plays a pivotal role in DR pathogenesis. By upregulating various inflammatory factors, phagocytic monocytes/macrophages and microglial cells are recruited, which in turn release various cytotoxic cytokines , 6. Cytoin vitro [In addition to inflammatory processes neovascularization is the main hallmark of DR pathogenesis and VEGF is postulated to be a key regulator of PDR . In addiin vitro  and dosein vitro , suggestin vitro –20. Therin vitro  and seemIn addition to Angiopoietins, Galectin-1 also plays an important and regulatory role in the proper execution of the angiogenesis process, such that loss of endogenous Galectin-1 in endothelial cells leads to impaired angiogenesis –24. ReceSeveral potential DR therapies have been investigated in recent years. By now, anti-VEGF therapy has become the first-line therapy. However, many treatments are often required over months and years to achieve successful treatment. Moreover, in about 30% of DR patients it can be observed that they do not respond to initial anti-VEGF treatment , which uThis raised the question of what other factors are involved in the development of DR and to what extent protein changes occur during DR progression. Hence, the aim of our study was to analyze protein alterations in vitreous samples of patients with different stages of DR, more precisely with NPDR and PDR, in comparison to DM patients without DR and controls (patients with macular hole or macular pucker undergoing vitrectomy) to reach a better understanding of DR pathophysiology. The gained understanding can lead to the development of further additive treatment options and a more precise adjustment of therapies to the DR course or form, which would support an individual treatment of the disease and significantly improve the overall quality of life.All patients gave their written informed consent before the start of the study as previously described and the Declaration of Helsinki was observed , 26. AppPrior to sample collection, patients were divided in four groups based on the clinical examination. The classification was based on the modified Arlie-House classification, also used in the Early Treatment in Diabetic Retinopathy Studies (ETDRS): Controls, diabetes mellitus (DM), non-proliferative diabetic retinopathy (NPDR), and proliferative diabetic retinopathy (PDR). For the first three groups, there was an indication for vitrectomy other than diabetic retinopathy, for example macular hole or macular pucker. For every patient clinical data, including mean age, gender, eye, DR stadium, fasting glucose, as well as HbA1c, were collected . PatientThe study originally included 135 patients (27 patients per group). However, vitreous samples that were too small for the planned ELISA analysis were excluded. Therefore, vitreous samples (1 ml/patient) from 25 patients with DM, 12 patients with NPDR, and 21 patients with PDR were collected by transconjunctival pars plana vitrectomy using a 23-gauge cutter. For this purpose, a core vitrectomy was performed with the gauge cutter after insertion of the trocars and before turning on the infusion. A 10 ml microsyringe connected to the aspiration tube of the cutter was used to collect the vitreous sample. The samples were immediately frozen and stored at -80°C until analysis.All proteins in vitreous samples were quantified using commercially available enzyme-linked immunosorbent assay kits ELISA; . Each asAs in previous studies, a commercial predictive analysis program  was used to perform the statistical analyses . ANOVA fThe Pearson’s correlation coefficient (r) was calculated between patient’s individual protein levels and patient’s fasting glucose level or VEGF-A level.In total, 116 vitreous samples were analyzed, 61 from male and 55 from female patients. The patients in the control group had a mean age of 77.05±9.01 years and the DM patients a mean age of 80.11±6.98 years. The DR group was subdivided into NDPR and PDR patients. The NDPR patients had a mean age of 71.66±8.99 years and the PDR group of 62.26±14.42 years. Therefore, the control and DM patients were significantly older than the PDR patients (PDR vs. control or vs. DM: p<0.001).Regarding gender and side ratio of the operated eye no significant differences were detectable between all four groups (p>0.05).Moreover, laboratory values, which provide diabetic indications, were collected. The patients in the control group had a mean fasting glucose level of 77.05±9.01 mg/dl and a mean HbA1c value of 5.65±0.62. Both indicators were significantly higher in the DM, the NPDR, and the PDR group  in comparison to the controls  as well as to DM  and to NPDR samples , when compared to control  or DM samples . In comparison to the NPDR group no difference was detectable  from this study in contrast to the three other groups, namely controls , DM , and NPDR samples  in comparison to the control  and the NPDR  but not the DM patients  compared to the control group . In comparison to the DM  and the NPDR group  no differences were observable .In addition, the levels of the pro-inflammatory cytokines TNF-α and interferon gamma (INF-γ) were also analyzed but the expression level was under the detection level in all samples.The inflammatory chemokine (C-C motif) ligand 3 (CCL3) was detctable in all vitreous samples but the expression level, which is regulated by TNF-α and IFN-γ, showed no differences between the control group, the DM, the NPDR, and the PDR group .2 = 0.134; 2 = 0.035; Correlation analysis of protein concentrations of several markers in vitreous samples and fasting glucose levels were performed. A significant correlation between levels of VEGF-A and fasting glucose was noted for all samples (r = 0.366; p<0.001; r2 = 0.076; 2 = 0.121; Analyzing the correlation between the angiopoietin members and the fasting glucose level revealed a significant correlation between Angiopoietin-1 and the fasting glucose value for all samples (r = 0.328; p<0.001; r2 = 0.001; 2 = 0.140; In contrast, no significant correlation was found between patients individual IL-1β level and fasting glucose levels (r = 0.023; p = 0.813; r2 = 0.010, Furthermore, no significant correlation was found between the individual Galectin-1 concentrations and fasting glucose levels (r = 0.099; p = 0.386; r2 = 0.060; We additionally evaluated whether the level of Angiopoietin-1 level correlates with the level of Angiopoietin-2. We observed a significant correlation between both angiogenic factors (r = 0.245; p = 0.011; rIn addition to the correlation analysis of the fasting glucose level and the protein concentrations in the vitreous, correlation analyses between VEGF-A as a key marker for DR and the other proteins were also carried out.2 = 0.052; 2 = 0.121; 2 = 0.229; 2 = 0.074; 2 = 0.138; 2 = 0.039; A significant correlation between levels of Angiopoietin-1 and VEGF-A (r = 0.228; p = 0.023; rDR is a sight-threatening condition that is set to increase worldwide over the next few decades due to the aging of society and the increase in DM. Anti-VEGF therapies are applied in case of PDR or diabetic macular edema, but frequent administration is required, and the macula edema of some patients does not respond to the various available anti VEGF therapies resulting in significant visual loss despite treatment , 33.However, other candidates besides VEGF might contribute to disease progression. This led to the objective of this prospective study, which investigated which other factors are involved in the pathogenesis of DR and to what extent protein changes occur during DR progression. Therefore, in addition to VEGF, the level of other angiogenic factors and inflammatory cytokines in the aqueous humour between NDPR and PDR patients was directly compared in the present study.Therefore, we evaluated protein alterations during different stages of DR of 116 patients undergoing pars plana vitrectomy. The control and DM patients were significantly older than the PDR patients. The acquisition of patients undergoing pars plana vitrectomy for indications other than DR may result in an age difference between the groups. Using patients who undergo a pars plana vitrectomy as a control group is an established approach , 34, 35,Our study data showed an up-regulation of VEGF-A in the group of PDR compared to other groups. In addition, the VEGF-A concentration was positively correlated with fasting glucose level. This result agrees with the past studies of VEGF concentrations in the fluids of the eye and the known role of VEGF in PDR . The sucHowever, not only VEGF-A, but also the other examined angiogenic factors showed this up-regulation in the PDR group compared to the other groups. Thus, PIGF was significantly up-regulated in the PDR group and positively correlated with VEGF-A levels. This result is consistent with our previous study  as well In addition to the VEGF family, recent studies revealed that the angiopoietin family also plays a role in regulating blood vessel sprouting and growing. In this context, Angiopoietin-1 and -2 seems to act agonistic and antagonistic . AngiopoThe role of faricimab as a simultaneous inhibitor of Angiopoietin-2 and VEGF-A to treat diabetic macula edema is now established, and a commercial preparation is available . The farOn the other hand, nesvacumab, an Angiopoeitin-2 inhibitor, in combination with aflibercept revealed no vison improvement for patients with diabetic macula oedema in comparison to VEGF/PIGF inhibition monotherapy by aflibercept . TherefoSimilar to the angiogenesis factors, we could find an upregulation of pro-inflammatory cytokines. It should be noted that IL-1β values were generally very low and close detection limit of the ELISA kits and in the PDR group near the detection limit. Nevertheless, this result is consistent with our previous study, in which we did not differentiate between NDPR and PDR patients . In addiMoreover, we could detect an up-regulation of IL-8 in comparison to the controls and the NPDR, but not the DM group, which in this case is most likely due to the strong scatter in the DM group. Moreover, for the first time we could find that the individual IL-8 concentrations significantly correlate with the fasting glucose levels. The up-regulation of IL-8 in vitreous PDR patients has already been mentioned , includiRegarding the pro-inflammatory cytokines TNF-α and INF-γ, no expression was detectable with the used ELISA kits. There are contradictory statements in the literature regarding the importance of TNF-α as a vitreous marker for PDR. It was initially considered a marker , 50, butFurthermore, the expression levels of the inflammatory chemokine CCL3 showed no differences between the four groups. This is consistent with the fact that TNF-α and INF-γ expression is not altered, since the expression of CCL3 is regulated by TNF-α and IFN-γ , 52.Galectin-1 is responsible for the proper execution of the angiogenesis process also by activating the VEGFR-1 , 24, 53.Thus, our study indicates pro-inflammatory cytokines as well as angiogenetic factors, which contribute to the pathogenesis of DR. Interestingly all analyzed proteins revealed alterations in PDR but not in NPDR patients. Hence, additional identifications of protein alterations in DR patients should be performed to identify NPDR specific alterations. For the PDR treatment in the future, both anti-inflammatory and anti-neovascularization agents could be used, possibly simultaneously. Therefore, the role of Angiopoietin-1 and Galectin-1 should be analyzed in more detail.Our study demonstrates the importance of angiogenic factors, besides VEGF, in the development of PDR. Angiopoietin-1 and -2 as well as the new marker Galectin-1 seem to represent an interesting starting point for further treatment development for DR. Furthermore, we were able to confirm an increase in the pro-inflammatory biomarker IL-8 in patients with PDR. Interestingly, all investigated pro-inflammatory factors were not elevated in the NPDR patients. The results highlight new potential avenues for targeted or additive therapies that can help to identify and treat severe complications of DR to save and improve vision.


To prevent nosocomial transmission of SARS-CoV-2, infection prevention control (IPC) measures are implemented for patients with symptoms compatible with COVID-19 until reliable test results are available. This delays admission to the most appropriate ward based on the medical condition. SARS-CoV-2 rapid antigen detection (RAD) tests and point-of-care (POC) rapid RT-PCR (VitaPCR) were introduced at emergency department (ED) at Skåne University Hospital, Sweden in late 2020, but the consequence on patient flow and targeted admission is unknown.Patients presenting at the emergency department of a referral hospital (N = 2940) between 13-Nov-2020 and 12-Jan-2021 were included. The study period was delimited into three periods by the introduction of RAD tests and the VitaPCR. Participant data was collected from hospital records, and outcome variables were Length-of-Stay (LoS), intrahospital transfers and targeted admission to COVID-19 ward.Compared to baseline (RT-PCR only), RAD tests reduced ED Length-of-Stay (LoS) for participants with positive tests. Negative VitaPCR results reduced mean hospital LoS by 1.5 (95% CI 0.3–2.7) days and admissions to COVID-19 wards from 34.5 (95% CI 28.9–40.5) to 14.7 (95% CI 11.1–19.1) per 100 admissions and reduced transfers between hospital wards in the first 5 days from 50.0 (95% CI 45.0–55.0) to 34.0 (95% CI 30.3–37.9) per 100 admissions.RAD tests enabled prompt detection of SARS-CoV-2 infection which had pronounced effects on LoS at the ED. Negative VitaPCR enabled cessation of IPC measures and a negative test was associated with increased targeted admissions, reduced intrahospital transfers and shorter LoS at the hospital.The online version contains supplementary material available at 10.1186/s12879-022-07497-x. The coronavirus disease 2019 (COVID-19) pandemic is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged in China in late 2019 . Real-tiIn late 2020, the optimized point-of-care (POC) RT-PCR VitaPCR SARS-CoV-2 Assay  was introduced and implemented at the Skåne University Hospital, Lund, Sweden. The assay utilizes a single tube for collection of the nasopharyngeal swab, cell lysis and nucleotide extraction. The total analysis time is about 20 min. Sample preparation does not require specialized laboratory setting and the reported sensitivity and specificity for SARS-CoV-2 is 99.3% and 94.7%, respectively . This shDuring 2020, substantial reorganizations were made at Emergency Departments (ED) and hospital wards globally to cope with the extraordinary requirements caused by the SARS-CoV-2 pandemic, including infection prevention and control (IPC) precautions to prevent secondary cases among patients and hospital staff. The study region was largely affected by the second wave of the pandemic with a considerable increase of COVID-19 cases from 1 Sept 2020 to 31 January 2021 . Before Although the sensitivity and specificity of the VitaPCR is superior to that of any RAD test, and time to result is substantially shorter for VitaPCR than for RT-PCR tests analyzed at the core hospital laboratory, the effects of these improvements on patient care are unknown. We hypothesized that introduction of the faster tests in the algorithm facilitated clinical decisions at the ED, limited IPC precautions to when necessary and improved targeted admission. This study evaluates the introduction of RAD tests and VitaPCR based on length-of-stay at the ED and hospital ward, intrahospital transfers the first 5 days and targeted admissions to COVID-19 ward during the peak of the second wave.This retrospective observational study is based on data from patients presenting at the Emergency Department, Skåne University Hospital, Lund, Sweden between Nov 13, 2020, and Jan 12, 2021. The hospital is a regional referral center, but the ED primarily serves the population of Lund and near surroundings . The total annual ED visits in 2020 were 59,000 patients. The present study was divided into three distinct time periods separated by the dates for introduction of RAD tests and VitaPCR, respectively: Period 1 (Nov 13–Dec 2), Period 2 (Dec 3–Dec 22) and Period 3 (Dec 23–Jan 12). The standard of care from the beginning of the pandemic and Period 1 of the present study was analysis of nasopharyngeal samples for SARS-CoV-2 with RT-PCR at a core hospital laboratory facility . On Dec 3, 2020 (Period 2 of this study), POC rapid SARS-CoV-2 antigen detection test  was introduced together with an algorithm to select which analysis method that was to be used. On Dec 23, POC testing with VitaPCR was added to the algorithm. The algorithms are presented in Fig. Data on visits to the ED and hospital ward admissions were collected from the hospital records using key word search, as specified below.The hospital records for all adult patients (≥ 18 years) that presented at the ED during the study period were screened for inclusion in the study. Inclusion criterions were any of the following: known COVID-19 infection at presentation to the ED, treatment in isolation room in the ED, emergency alerts labeled “Infection”, or emergency alerts with main complaints marked as “dyspnea”, “fever”, “infection”, “confusion”, “shock”, “cardiac arrest” and “non-specified illness”. All eligible patients during the study period were included.SARS-CoV-2 test results and test methods were recorded and used for grouping of participants into independent groups: “Positive test at the ED” and “Negative test at the ED” included participants for which a nasopharynx test was taken at the ED and analyzed for SARS-CoV-2 by RT-PCR, RAD test or VitaPCR. For RAD test and VitaPCR, the test result was available before the patient left the ED, while this was not the case for RT-PCR. Participants with positive SARS-CoV-2 analyzed before presentation to the ED were not sampled for SARS-CoV-2 and here labelled as “Positive test before admission”. Finally, “Not tested” included participants not tested for SARS-CoV-2 at the ED and the medical records did not specify COVID-19 status. The ICD-10 diagnoses at discharge from the ED and hospital wards were recorded and aggregated into compound variables  or 95% confidence intervals (CI). Categorical variables are presented as counts, fractions of total and 95% CI calculated with the Wilson/Brown method to allow for comparisons between groups (overlapping ranges represent insignificant differences). Comparisons were also evaluated by One-Way-ANOVA with Tukey’s multiple comparison tests and by Fisher’s exact test. Analyses were performed with GraphPad Prism 9.0 . P-values < 0.05 were considered statistically significant. Missing data is presented in the tables.A total of 9325 patients visited the Emergency Department during the study period and 2940 of these met the inclusion criteria and were selected for enrollment in the study. There was a consecutive increase in the total number of patients that visited the ED and an increase in the proportion that met the inclusion criteria: In Period 1: 781 participants out of 3024 patient visits (25.8%) met the criteria, in Period 2: 988 participants out of 3149 patient visits (31.4%), and Period 3: 1171 participants out of 3152 patient visits (37.2%). The mean age was 60.8 (SD ± 20.8) years and 1497 (50.9%) of the participants were women.During the study period, a total of 1866 (63.5%) participants were tested for COVID-19 at the ED. There was no significant difference in testing percentage between men and women (48.3% vs 51.7%). The mean age among participants that were tested for SARS-CoV-2 was 64.1 (SD ± 20.0) years and the mean age among those not tested was 55.1 (SD ± 21.0) years. As the study periods were defined by changes in testing routines, substantial differences in SARS-CoV-2 testing methods were observed between the periods  tested positive for SARS-CoV-2 infection at the ED, 1458 (49.6%) tested negative, 568 (19.0%) had had a positive test before admission to the ED, and 506 (17.2%) were not tested for SARS-CoV-2 infection at the ED (Table The mean Length-of-Stay (LoS) at the ED was 374 (SD ± 269) minutes , to 14.8% (95% CI 12.7–17.1) in Period 2 and 19.6% (95% CI 17.4–21.9) in Period 3. No statistically significant changes were seen for other diagnoses.Of the 2940 participants, 1487 (50.6%) were admitted to a hospital ward and 1453 (49.4%) discharged to home. For the entire study population, the fraction of participants that were discharged to home from the ED did not change notably during the study period. However, a larger fraction of the participants that were tested for SARS-CoV-2 could be discharged to home from the ED after introduction of RAD tests and VitaPCR  days Table . The meaThe overall proportions of participants that were admitted to COVID-19 wards, Mixed COVID-19/Internal medicine wards, ICU or Other wards did not vary significantly during the study period Table . HoweverSimilarly, participants with “Negative test at the ED”, were 57% less likely to be admitted to a COVID-19 ward and 81% more likely to be admitted to a non-COVID-19 hospital ward after the introduction of both the RAD test and VitaPCR  in Period 1, introduction of RAD test in Period 2 and introduction of VitaPCR in Period 3. Importantly, the results reveal that the implementation had a significant effect on length-of-stay at the ED and hospital, intrahospital transfers first 5 days and targeted admission to wards with IPC facilities, which has implications for the treatment of the individual patient, patient safety, hospital infection control and optimal resource use.Before the introduction of point-of-care rapid SARS-CoV-2 tests at the ED, patients with suspected COVID-19 infection were admitted to COVID-19 diagnostic and treatment wards with IPC facilities until results from RT-PCR became available. Patients with negative tests were then transferred to the most appropriate ward based on the medical need. While this prevented secondary cases of COVID-19, intrahospital transfers have been related to unfavorable events such as increased falls, medication errors, length-of-stay and hospital-acquired infections , 12, as No significant effect on targeted admission variables could be seen for test positive participants, likely because both suspected and confirmed cases of COVID-19 were admitted to wards with IPC measures in all study periods. However, hospital LoS for patients that had a positive SARS-CoV-2 test taken before admission to the ED was longer in Period 2 and 3 compared to the first period of the study. The reason for this is not known. No difference in age or in proportion that received ICU care (data n.s.) were seen between the periods.High patient load at the ED puts increased pressure on hospital wards, which can lead to ED crowding, extended LoS and decreased patient safety . The oveThe fraction of patients that were discharged from the ED to home increased during the study period. This increase was seen after introduction of the RAD tests, possibly because of the faster diagnostic work up that was enabled by the rapid tests. In the algorithms used during the study period, a positive RAD test was enough to confirm COVID-19. Hence, it can be expected that any change in patient care inflicted by the rapid tests for participants that tested positive at the ED would occur already in Period 2 (after introduction of RAD test) and not change significantly after introduction of the VitaPCR.The study period is unique in that allows for interrogation of the sequential introduction of two rapid POC analysis methods with direct implications for IPC management of patients in a high endemic setting. The study site is the only ED in the area and the hospital treats all kinds of medical emergencies, which resulted in an unbiased adult population that is generalizable to similar ED’s. The study population was sufficiently large for the proposed analysis. The conclusions drawn here may well be applicable in other similar settings, for instance the need for rapid and accurate diagnostic tools for the annual influenza virus epidemic, which put similar demands on ED and hospital wards. However, during the COVID-19 pandemic different strategies to prevent nosocomial transmission have appeared and testing strategies have evolved depending on the availability of RAD and RT-PCR tests. The effect of introducing a new test on patient flow is dependent on the setting and timing of when the test is taken. An additional test can have negative consequences on patient flow, for instance when required for transfer between hospital and nursing home .Limitations of the present study mainly relate to the retrospective study design. Data collection was limited to data available in hospital records. Although the only known difference between the study periods was the testing routines, confounding factors cannot be excluded. We can only describe differences between the periods that are associated to the introduction of respective test, and not state any cause-effect. These potential biases could have been avoided with a prospective study design in which participants were randomized to RT-PCR, RAD-test or VitaPCR. At the time of introduction of these diagnostic methods, it was not possible, or ethically defendable, to conduct a prospective randomized study.In conclusion, the implementation of rapid tests for SARS-CoV-2 and an algorithm that included VitaPCR was for test-negative participants associated with an increase in targeted admissions to an appropriate ward, reduced intrahospital transfers and shortened LoS at hospital wards. For positive patients, this change was associated with shorter LoS at the ED and observed already at introduction of RAD tests. It would be of great interest to further investigate the health-economic implications of these results.Additional file 1. Grouping of ICD-10 diagnosis on discharge from the ED and hospital wards into compound variables.


In fire accidents, highly toxic gases such as carbon monoxide (CO) and hydrogen cyanide (HCN) are simultaneously generated during the combustion of building materials. When inhaled, these two gases strongly bind to hemoglobin, cytochromes, and other hemes in the living organisms, thus inhibiting aerobic respiration. To date, there is no therapeutic approach to overcome simultaneous poisoning with CO and HCN. Here, we invented a synthetic heme model compound (hemoCD-Twins) to provide emergency life-saving treatment. This compound captures CO and cyanide inhaled into animals with a single injection and is rapidly eliminated via urinary excretion. With an immediate antidotal effect, high degree of safety, and storage stability, hemoCD-Twins has great potential to be a ready-to-use antidote against fire gas poisoning. –) mixed poisoning. The solution contains four compounds: iron(III)porphyrin , two methyl-β-cyclodextrin (CD) dimers linked by pyridine  and imidazole , and a reducing agent . When these compounds are dissolved in saline, the solution contains two synthetic heme models including a complex of F with P (hemoCD-P) and another one of F with I (hemoCD-I), both in their iron(II) state. hemoCD-P is stable in its iron(II) state and captures CO more strongly than native hemoproteins, while hemoCD-I is readily autoxidized to its iron(III) state to scavenge CN– once injected into blood circulation. The mixed solution (hemoCD-Twins) exhibited remarkable protective effects against acute CO and CN– mixed poisoning in mice . In a model using rats, exposure to CO and CN– resulted in a significant decrease in heart rate and blood pressure, which were restored by hemoCD-Twins in association with decreased CO and CN– levels in blood. Pharmacokinetic data revealed a fast urinary excretion of hemoCD-Twins with an elimination half-life of 47 min. Finally, to simulate a fire accident and translate our findings to a real-life scenario, we confirmed that combustion gas from acrylic cloth caused severe toxicity to mice and that injection of hemoCD-Twins significantly improved the survival rate, leading to a rapid recovery from the physical incapacitation.Simultaneous poisoning by carbon monoxide (CO) and hydrogen cyanide is the major cause of mortality in fire gas accidents. Here, we report on the invention of an injectable antidote against CO and cyanide (CN Their amounts depend on the materials of combustion (52) transport/storage in the blood circulation and tissues. On the contrary, HCN binds as cyanide ion (CN–) to ferric iron(III) heme, preferentially targeting cytochrome c oxidase (CcO) in mitochondria and interrupting O2-dependent energy production porphyrin and two cyclodextrin (CD) dimers. Based on our previous research on the synthesis and characterization of heme protein model structures (Left) is stable in the iron(II) state in vivo and shows much higher CO-binding affinity compared to the affinities reported for native heme proteins (Right) is stable in the iron(III) state and shows a higher binding affinity to CN– than native ferric met-hemoglobin (met-Hb) (– to their iron(II/III) centers in vivo and expel these toxic ligands from the organism  The synthetic compounds are storable at room temperature over a year thanks to their chemical stability; 2) the solution can be quickly prepared without the need of special handlings; 3) a single administration of hemoCD-Twins shows immediate dual antidotal effect against CO and CN–; and 4) the injected solution is quickly eliminated from the body without significant side effects. We are envisioning a scenario whereby a person who is accidentally exposed to fire gases containing CO and HCN can be promptly treated at the site by infusion with hemoCD-Twins.In the present study, we have developed an injectable antidote for CO and HCN mixed poisoning. This agent contains supramolecular compounds, termed ructures –40, the ructures A, Left iproteins –45. On tproteins A, Right (met-Hb) , 47. Theorganism –47. HereO-methylated β-CD dimers having pyridine (Py3CD or P) and imidazole linkers (Im3CD or I), and sodium dithionite (S) in phosphate-buffered saline (PBS) (SI Appendix). These four compounds are stable in powder and storable at room temperature. While S is commercially available, F, P, and I are synthesized in our laboratory . Solutions containing 3 to 14 mM of F were used in this study, which were readily reduced to iron(II) by adding excess S . The mixed solution of F, P, I, and S, i.e., the mixture of hemoCD-P and hemoCD-I in the iron(II) states, is hemoCD-Twins that is ready for injection to scavenge CO and CN– simultaneously in vivo porphinatoiron(II/III) (FeTPPS or F), per-ne (PBS) B  in the stock solution was rapidly consumed by O2 and dissolved in the buffer to be converted to sulfate (SO42–) and H2O with superoxide and hydrogen peroxide as intermediates  state, i.e., autoxidation  and hemoCD-I , respectively . Therefore, autoxidation of hemoCD-I is much faster than that of hemoCD-P, and the two compounds autoxidized independently even in their mixed system, i.e., hemoCD-Twins. The ferrous hemoCD-P bounds CO and ferric hemoCD-I bounds CN– in solution. Indeed, hemoCD-Twins in air-saturated PBS showed dual reactivity to both CO and NaCN independent from their addition order . The ligand-binding affinities and kinetic parameters for hemoCD-P and hemoCD-I have been measured previously .When the solution of hemoCD-Twins  was diluted in air-saturated PBS (3 mL), excess dithionite  were intraperitoneally (i.p.) administered to mice.In this study, unanesthetized mice   were useInset). The absorption spectra of the urine solutions confirmed the excretion of these compounds (t1/2 = 36 to 39 min in vitro) might be accelerated in vivo (88% autoxidized in 30 min), probably due to difference in the two biological milieux. The autoxidation rate of the oxy–iron(II) complexes in H2O is significantly influenced by coexisting anions as demonstrated previously by Shikama  of hemoCD-P , hemoCD-I , and hemoCD-Twins  with 5 mg/mL S (29 mM) in healthy conscious mice, brownish-red–colored urines were naturally excreted within 30 min . Histopathological data showed no significant abnormalities in these tissues after administration of hemoCD-Twins . Moreover, there were no significant changes in biochemical markers of kidney and liver functions  compared to controls treated with PBS . These results confirm the safety of hemoCD-Twins in mice. To exclude a possible toxicity of the reducing agent sodium dithionite (S), mice were challenged with this compound at various doses administered i.p. . The dose of 1,000 mg/kg S was found to be lethal. The dose of S present in the complex mixture of hemoCD-Twins (5 mg/mL in 0.2 mL per mouse of 20 ± 2 g) calculated at 50 mg/kg is 20 times lower than the toxic dose of S alone observed in mice.We also found that 24 h after treatment with hemoCD-Twins, all the mice showed no difference in behavior compared to controls and no residual porphyrin compound was visually observed in major organs  . While 10,000 ppm CO caused significant lethality (86%), 7,500 and 5,000 ppm CO were less toxic, inducing 33 and 0% lethality, respectively. Indeed, these two lower doses were associated with a gradual recovery of mice from their physical incapacitation . Similarly, the effects of oral administration of NaCN  dissolved in PBS (0.1 mL) were also tested , and a significant lethality was found at 0.20 mg NaCN. Notably, while most mice survived when independently challenged with either 0.15 mg NaCN or 5,000 ppm CO, a rapid lethal toxicity was observed within 40 min when these two compounds were administered simultaneously (– (Left), resulting in approximately 85% of mice surviving 24 h after treatment (n = 11/13). Blood carboxyhemoglobin (CO-Hb) and CN– levels in mice reached a maximum of 51% and 2.1 µg/mL, respectively. These values decreased markedly to 8.8% and 0.31 µg/mL, respectively, 45 min after administration of hemoCD-Twins . In addition, hemoCD-Twins-treated mice recovered from their physical incapacitation within 15 min after injection (Right); that is, mice that became unconscious due to the simultaneous poisoning by CO and CN– regained their mobility and began to walk within a short time after administration of hemoCD-Twins. We note that treatment with either hemoCD-P or hemoCD-I after combined exposure of mice to CO and NaCN also showed significant effects in reducing lethality and enhancing the behavioral recovery, although the recovery rates were slower and less pronounced than those of mice receiving the dual antidote system, hemoCD-Twins . In fact, lactate concentration increased from 5.9 ± 1.5 mM to 19.2 ± 3.1 mM at 6 min after combined exposure to CO and CN– and significantly decreased to 6.5 ± 1.1, 12.3 ± 1.1, and 16.8 ± 4.1 mM at 39 min (t45) after injections of hemoCD-Twins, hemoCD-P, and hemoCD-I, respectively. Therefore, hemoCD-Twins exerted a marked therapeutic effect on lactic acidosis induced by CO and CN– . When hemoCD-P or hemoCD-I at the doses of 1.0, 3.5, or 7.0 mM was injected into mice intoxicated with 5,000 ppm CO or 0.1 mg NaCN, these compounds were excreted in urine bound to their respective ligands with different degrees of saturation. While at the lower doses the complexes were almost saturated by these ligands, we measured 53% saturation of hemoCD-P with CO and 67% saturation of hemoCD-I with CN– at the highest dose used. Thus, by injecting a dose of 7 mM, there is still enough compound in the circulation with the capacity to bind additional CO or CN–. These results demonstrate that the dose of 7 mM hemoCD-P or hemoCD-I  is sufficient to treat mice and neutralize the CO/CN– mixed poisoning used in our protocol.A better recovery after hemoCD-Twins treatment was also evident when looking at the lactate levels in blood . During the combustion phase, the gas components in the mice chamber were quantitatively analyzed by a Fourier transform infrared spectroscopy (FT-IR)  was administered. As shown in To further test the therapeutic effects of hemoCD-Twins in a real-life scenario, combustion gas from acrylic cloth was used to poison mice in a combustion gas toxicity tester ( (FT-IR) , reveali (FT-IR) B. The gaMale rats  were anesthetized with isoflurane and used to assess the pharmacokinetic of hemoCD-Twins and its effect on hemodynamic parameters. The toxicological effects of CO and HCN in this model are assumed to be essentially the same as the ones produced in mice, with no particular relevance about differences in species or sex.SI Appendix, Fig. S13). The plasma concentration of hemoCD-Twins increased immediately and then rapidly decreased after infusion (ca. 98% of the injected hemoCD-Twins was excreted within 2 h (ca. 2.8 nm) of the complex, its hydrophilicity, and little interaction with plasma proteins as we have previously determined when studying the pharmacokinetics of hemoCD-P , indicating that the infused hemoCD-P and hemoCD-I were eliminated via renal clearance at similar rates. Rapid infusion (12 mL/h) of hemoCD-Twins to healthy rats showed no change in their heart rate and a slight increase in mean blood pressure . Biochemical markers  indicative of renal and liver functions assessed in rat plasma before and 3 h after infusion  revealed small effects of hemoCD-Twins on these parameters except for BUN. The increase in BUN (from 24.0 to 38.6 mg/dL) is probably due to the presence of some residual hemoCD-Twins still present in the body 3 h after administration.A PBS solution (2 mL) of hemoCD-Twins (14 mM) or its controls  was intravenously (i.v.) infused into the femoral vein of anesthetized rats. After starting the infusion of 2 mL of hemoCD-Twins at the rate of 12 mL/h , blood plasma samples were collected at different time points and the amount of hemoCD-Twins was quantified by size exclusion chromatography , hemoCD-P elicited a more rapid decrease in CO-Hb% than hemoCD-I. In contrast, hemoCD-I markedly reduced CN– levels (– poisoning and 2) the antidote activity of hemoCD-Twins is superior to that of hemoCD-P and hemoCD-I when used alone.Successive additions of NaCN  and CO gas  to the anesthetized rats significantly reduced mean arterial pressure and heart rate as shown in  or NaCN C and D. – levels D while h–. In addition, the compound is readily excreted in urine and totally disappears from the body within few hours. Using various experimental models, we show that hemoCD-Twins significantly protects mice and rats from poisoning and death caused by the combination of CO and CN– exposure or by combustion gas produced during the burning of acrylic cloth. Thus, hemoCD-Twins is a stable and urinary-excretable compound that functions as an antidote against CO and CN– in vivo.In the case of fire accidents and intoxication by combustion gas in humans, a storable, ready-to-use, quick-acting antidote would be highly desirable for in-situ treatment. In this study, we report on the invention of such antidote substance, hemoCD-Twins, using biomimetic heme protein model complexes. The components of hemoCD-Twins are stable for over one year with no sign of decomposition when stored in the ferric iron(III) solution. When injected, ferrous hemoCD-P captures CO, while ferrous hemoCD-I is readily oxidized to its ferric iron(III) form and captures CN– should have high relevance in the context of fire gas poisoning, but this issue has not been addressed experimentally.As it is well recognized, CO poisoning frequently occurs in fire accidents. Generally, victims who die because of CO poisoning tend to exhibit over 60% CO-Hb in their blood. On the contrary, death in subjects with less than 30% CO-Hb has been also frequently reported , 59. One– antidotes. For CO poisoning, normobaric/hyperbaric O2 ventilations are practically applied; this intervention requires transportation of patients to hospitals equipped with O2 cylinders and normobaric/hyperbaric chambers, causing significant time delays (H64Q-CCC) showed much higher CO binding affinity than Hb in red blood cells and thus rapidly decreased CO-Hb levels and improved survival rate when administered to mice exposed to CO. The functional mechanism of hemoCD-P as a CO antidote is basically the same as that of the neuroglobin mutant. The high CO-binding affinity and the CO/O2 selectively of hemoCD-P make this complex able to remove CO from native heme proteins with high efficiency . Moreover, the compound shows a slow autoxidation of the iron(II) complexes, satisfying the requirement for an in vivo CO scavenging molecule. Accordingly, our group has recently shown that an intravenous infusion of hemoCD-P to rats exposed to toxic CO levels promptly reduces CO bound to Hb and accumulation of CO in organs and tissues including the brain  are currently adopted as therapeutic interventions . However, treatment with nitrite compounds causes significant disruption of O2 transport by ferrous Hb in red blood cells and thus it is not an ideal compound to be used in the case of fire accidents where CO intoxication is possibly concurrent   center of OHCbl  . Additioof OHCbl . Compare albumin .– poisoning. The antidotal effect of hemoCD-Twins on CO and CN– mixed poisoning was remarkable in the mice and rat models examined in this study. We used conscious mice for survival tests using a gas-tight chamber (4 L) and injected them intraperitoneally with hemoCD-Twins for quick intervention. In addition, we used anesthetized rats for pharmacokinetic and hemodynamic studies because of the ease of surgery and intravenous administration of the compounds. We observed that the toxic effects of CO and CN– are basically the same in mice and rats; therefore, the results obtained from these two rodent species are discussed here without any distinction. Because of a high transfer efficiency from the abdominal cavity to blood (>50% for hemoCD-P)  , both i.emoCD-P) , 48. IndhemoCDs in its reduced state. We have previously used hemoCD-P in vivo after removing excess S by passing the reduced complex through a desalting column  , and we confirmed in our experiments a lethal effect of S at 1,000 mg/kg following i.p. injection in mice . The amount of S contained in hemoCD-Twins is 50 mg/kg (29 mM), far below the reported toxic levels. In addition, approximately half a mole of dithionite (14 mM) is consumed to reduce hemoCD-Twins before injection. Therefore, the actual dose of dithionite administered would be below 25 mg/kg, which is much lower than the toxic one. Indeed, hemodynamic, histopathological, and biochemical analyses of healthy rodents treated with hemoCD-Twins at the effective doses  showed negligible effects. Among the additional reducing agents we have tested including ascorbate, sulfite, metabisulfite, and methylene blue, dithionite (S) appears to be the most efficient one to rapidly and quantitatively form the ferrous iron(II)porphyrin complexes. We understand that further safety tests and optimization of our antidote formulation would be necessary for clinical use of S as a reducing agent, but the data presented here appear promising. Another important issue to consider is that once in the circulation, hemoCD-I in the reduced state can be readily autoxidized and this may lead to the production of superoxide anion (O2 + Fe(II) → Fe(III) + O2•–). The influence of superoxide and other related reactive O2 species has not been investigated here, but the presence of small amount of superoxide dismutase and catalase as adjuvants to the hemoCD-Twins formulation might be considered in future studies.Concerning the safety of new compounds, we have repeatedly assessed that both hemoCD-P and hemoCD-I show little cytotoxicity, hemolytic activity, and marginal effects on hemodynamic parameters upon their administration to mice and rats , 47, 64.g column , 44, 65.tration)  comparab– concentrations in blood is quite time-consuming (as we experienced in rat blood tests), perhaps explaining why CN– intoxication has been often ignored in the diagnosis. Indeed, in forensic anatomy of fire-related accidents from 2007 to 2009 in Japan . Mixed intoxication of CO and HCN is a major and often-overlooked factor in the case of building/urban fires. We envisage that hemoCD-Twins can be quickly administered to patients suffering from fire gas exposure without the need to wait for the diagnosis of whether patients are primarily intoxicated by CO and/or HCN. Additionally, hemoCD-Twins has the potential to act as an antidote to other toxic gases such as nitric oxide and hydrogen sulfide that can also bind to heme-related proteins, which are currently being investigated in our laboratory , while measuring CNin Japan , measureboratory , 69. Fin– mixed poisoning. The biochemical and preclinical studies presented here indicate promising therapeutic features of this compound based on its redox properties, pharmacokinetic behavior, minimal side effects, and effective scavenging activities against CO and CN–. Future tests in large mammals and humans will provide definite and conclusive evidence for the efficacy of hemoCD-Twins as a breakthrough antidote that might save human lives from frequently occurring gas poisoning episodes worldwide.In conclusion, we have developed hemoCD-Twins as an injectable and urinary-excretable antidote against CO and CNIIITPPS (F), Py3CD (P), and Im3CD (I) were synthesized in our laboratory. The synthetic procedures were previously reported  solution. Before injection to animals, the ferric iron(III) solution was reduced by mixing with 5 mg/mL S to give the ferrous iron(II) solution. Autoxidation reaction from the iron(II) to iron(III) complexes was monitored by UV-vis spectroscopy (Shimadzu UV-2450 spectrophotometer). Detailed procedures of sample preparation and chemical characterization are available in – intoxication models including the model using combustion gases, we used female BALB/cCrSlc mice. After the mice were exposed to these toxins, the solution of hemoCD-Twins in PBS (0.2 mL) was injected intraperitoneally. The mice were then monitored for survival rate and behavioral recovery under aerobic conditions at room temperature. For pharmacokinetic and hemodynamic studies, we used male Wistar rats (300 ± 15 g). Rats anesthetized with isoflurane were infused i.v. with hemoCD-Twins. During and after the infusion, heart rate and blood pressure were continuously monitored using Bio Amps (AD Instruments Ltd). Plasma and urine concentrations of hemoCD-Twins in rats were quantified by UV-vis spectroscopy (Shimadzu UV-2450 spectrophotometer) and size exclusion chromatography , respectively. A full description of these animal experiments is available in SI Appendix.All animal studies were performed under the approval of Doshisha University, Doshisha Women’s College of Liberal Arts, Tokai University, Building Research Institute, and Center for Better Living. For monitoring the therapeutic effect of hemoCD-Twins on lethal CO/CNt test. Survival curves were analyzed using Kaplan–Meier curves and log-rank test. Differences with P values of less than 0.05 were considered significant.Statistical analyses were performed using GraphPad Prism, Version 8.0 (GraphPad Software). All data represent the means ± SE from at least three different experiments and were analyzed by Student’s Appendix 01 (PDF)Click here for additional data file.


Acute myocardial infarction (AMI), commonly known as a heart attack, is a life-threatening condition that causes millions of deaths every year. In this study, a transistor-based biosensor is developed for rapid and sensitive detection of cardiac troponin-I (cTnI), a diagnostic biomarker of AMI. A biosensing technique based on a field effect transistor (FET), which uses indium gallium zinc oxide (IGZO) as an excellent semiconducting channel, is integrated with nanosheet materials to detect cTnI. Porous carbon nitride (PCN) decorated with gold nanoparticles (Au NPs) is used as a bridge between the solid-state device and the biorecognition element. We demonstrate that this biosensor is highly sensitive and has an experimental limit of detection of 0.0066 ng/mL and a dynamic range of 0.01 ng/mL–1000 ng/mL. This is the first report of a semiconducting metal oxide FET cardiac biomarker sensor combined with PCN for the detection of cTnI. The reported compact microsystem paves the way for rapid and inexpensive detection of cardiac biomarkers. Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide ,2. CVD mDS), are influenced by the reaction between the biomarker and the biorecognition element  CGT-GCAGTACGCCAACCT TTCTCATGCG CTGCCCCTTA (Tro4) is a thiol-modified Anti-cTnI DNA aptamer obtained from Sigma . Mercapt2SO4 and ultrasonically agitated until the yellow solution became colorless. The solution was then dispensed into 250 mL of ice-cold water to yield white-colored PCN. The product was separated and washed with water and ethanol. To prepare PCN-Au NPs, 0.5 g/mL aqueous PCN dispersion was first prepared in 50 mL of water to which 0.10 M HAuCl4 (1 mL) and 0.10 M NaBH4 (10 mL) were added and stirred for 1 h. Finally, the product was separated, washed, and dried to obtain PCN-Au NPs.GCN was prepared from melamine via a typical thermal polymerization procedure. One gram of melamine was placed in a silica crucible and annealed at 550 °C for 4 h (heating rate: 5 °C/min). The resulting powder was washed three times with deionized water (DI) and ethanol to obtain GCN. Next, 10 mg/mL GCN dispersion was prepared in 100 mL H2) with 150 nm thickness was deposited as a gate through a thermal oxidation procedure. Heavily doped n-type (n++) silicon wafers with thermally produced chlorinated SiO2 layers were employed to fabricate the bottom-gate bottom-contact FET devices. The wafer samples were cleaned using an ultrasonic cleaner for 5 min each in acetone and isopropyl alcohol (IPA), dehumidified for 5 min at 120 °C after being dipped in water, and briefly exposed to N2 gas. RF sputtering was utilized to deposit a thin film of IGZO with a thickness of 10 nm following our previously reported procedure [2 (20 SCCM/3 SCCM) plasma at a deposition pressure of 5 m Torr and 60 W RF power. Rapid thermal processing (RTP) at 5000 °C for 4 min in an O2 atmosphere was used to increase the stability of the FET device.IGZO FETs were fabricated via a bottom-up approach. The n++ Si wafers were procured from Silicon Materials (Si-Mat), and the device fabrication was carried out in the KAUST nanofabrication core labs. The silicon oxide that serves as the gate dielectric was grown using thermal oxidation, and IGZO was RF sputtered using a ceramic IGZO target. Organic and metal impurities were entirely eliminated from the Si wafers by dipping them in Piranha solution for 5 min. A native oxide etch was performed with buffered oxide etch solution. Then, silicon oxide  sputter equipment was used to deposit Ti (10 nm)/Au (100 nm) on top of the as-prepared substrate. After a reasonable duration of sonication in acetone, the source and drain IDEs were obtained.Lift-off deposition was utilized to deposit interdigitated top electrodes made of titanium (Ti) and gold (Au), then the interdigitated electrodes (IDEs) were fabricated by photolithography. DC magnetron sputtering at 400 W in the presence of Ar plasma was used to deposit metals, yielding Ti and Au with thicknesses of 10 nm and 100 nm, respectively. The IDE designs were fabricated using a typical photolithography procedure. The Si/SiOv/v), and 1.0 µL of PCN-Au NPs was drop-casted onto the surface of the FET-IGZO devices and dried at 6 °C for 15 min.The dispersion of PCN-Au NPs (1.0 mg/mL) was prepared in 50% water/ethanol  were prepared in 10 mM PBS (pH 7.40). The active channel of the aptasensors was coated with approximately 1.0 µL cTnI solution, incubated for 10 min to allow binding, and then the devices were cleaned with water to remove the unbound analyte.First, a mixture of 1 mM (optimized) cTnI aptamer and 20 mM MCH was prepared in 10 mM PBS (pH 7.4), then 1.0 µL of the aptamer-MCH mixture was dropped onto the active channel of the PCN-Au NP-modified FET-IGZO, incubated for 12 h, and the modified device was cleaned three times with water (pH 7.40). The device was subsequently treated with 0.10 mg/mL BSA for 30 min to prevent non-specific binding. The final device is a FET-IGZO PCN-Au NPs aptasensor. Various concentrations of cTnI  = −1.0 V to 20 V for various drain voltages (VDS) stepped from 5.0 V to 30 V, and the resultant transfer characteristics were measured. To measure the output characteristics, the drain voltage was scanned between 0 V and 8.0 V, and the resultant gate voltage was stepped at 8.0 V. The transfer and output properties of the device were measured at various concentrations of cTnI. All the analyses were performed at least three times, and the average of those results was utilized to make the figures.A commercial Keithely 4200 semiconductor characterization system (SCS) was used to measure the electrical behavior of the FET-IGZO PCN-Au devices in the presence of various concentrations of cTnI. The Bio-FET device was biased by scanning the gate voltage , -C-N-H, and C-N bonds with binding energies of 398.2, 398.9, 399.8, and 400.8 eV, respectively, were observed, validating the structure of PCN [7/2) and 87 eV (4f5/2), which demonstrates that Au was successfully loaded onto the PCN sheets, causing a localized fluctuation in PCN electron density and donation of electrons from PCN to the Au NPs [The synthesis route for PCN-Au NPs is shown in e of PCN . Additioe Au NPs . N-defec2 and contained the elements in IGZO, carbon, nitrogen, and gold (A schematic illustration of the PCN-Au NP-based FET-IGZO sensor with the cTnI aptamer for cTnI protein detection is shown in and gold e.gs (8 V) to investigate the performance of the sensor.A bottom-gate top-contact FET configuration was chosen to facilitate the functionalization of the top semiconducting IGZO channel with a biorecognition element. The output characteristics of the as-fabricated IGZO are displayed in DS responses of the PCN-Au NPs Bio-FET sensor gradually decreased. This decrease results from the specific binding of the aptamer to the cTnI, which hinders electron transport. The hindrance varies depending on the amount of cTnI bound to the Bio-FET. The linear range of the calibration curve, which was obtained at VDS = 6.50 V, was between 0.01 ng/mL and 1000 ng/mL , which covers the clinically significant range of cTnI in actual patient samples. The concentrations of cTnI range from 0 to 0.01 ng/mL in the blood of healthy patients, from 0.01 to 0.04 ng/mL in patients with mild myocarditis, and from 0.06 ng/mL or more in patients with necrotic myocardial injury [PCN-Au NPs Bio-FET was used to create an ultrasensitive cTnI sensor a. The PC00 ng/mL c. The coWe successfully demonstrate a PCN-Au NPs and IGZO Bio-FET sensor for rapid, sensitive, quantitative monitoring of cardiac troponin biomarkers (cTnI). PCN-Au NPs offer a highly porous and enhanced surface area for the immobilization of aptamers. The PCN and IGZO materials are compatible with transistor device format and offer significant biosensing properties in terms of accommodating and providing microstructures for cTnI aptamers and enhancing the binding effectiveness of the cTnI with the transistor. The biosensing features of the Bio-FET (linear range: 0.01–1000 ng/mL) and detection limit 0.0066 ng/mL) are in a clinically useful range, suggesting that the biosensor could be used for practical applications. This sensor holds potential for the rapid, sensitive diagnosis of AMI. Future research is directed toward translating this device into a point-of-care device that can be used in non-laboratory settings.